Uti fusion proteins

ABSTRACT

The present invention provides UTI fusion proteins, DNA sequences for producing the same, and pharmaceutical compositions and methods of using the same.

FIELD OF THE INVENTION

The present invention relates to molecular biology, pharmacology, andmedicine.

BACKGROUND OF THE INVENTION

Urinary Trypsin Inhibitor (UTI), also known as ulinastatin, uristatin,urinastatin, ulistin, human inhibitor 30 (HI-30), mingin and bikunin, isa protease inhibitor with a molecular weight of about 40 kD. UTI ispresent in human urine and blood (hUTI) and has a variety ofphysiological activities such as an inhibitory effect on a family ofserine proteases, such as trypsin, α-chymotrypsin, plasmin, cathepsin-Gand leukocyte elastase. UTI also has immunomodulatory effect, and it candown-regulate the release of proinflammatory cytokines, such as tumornecrosis factor-α (TNF-α), interleukin-1 (IL-1) and interleukin (IL-6).In addition, UTI also interferes with PDGF-D (PDGF-DD)/PDGF-BBR activedimer-mediated signaling pathway by neutralizing the dimer.

hUTI has received marketing authorization and one product is marketed inJapan under the trade name Miraclid and is isolated from human urine. Infact, hUTI isolated from human urine is currently marketed by severalmanufacturers for treatment of pancreatitis and acute circulatoryfailure caused by shock.

UTI is first produced in humans as a presursor protein called AMBP(al-microglobulin/bikunin precursor), which is encoded on humanchromosome 9. The proteolysis of AMBP yields the free UTI containing 143amino acids. UTI comprises two Kunitz domains that are known to inhibitserine proteases, which are flanked by unstructured amino acids on UTI'sN- and C-termini. The two domains are expected to confer differenctspecificities of protease inhibition, due to the different amino acidsinvolved in protease binding. By analogy to other serine proteaseinhibitors (e.g. BPTI, bovine pancreatic trypsin inhibitor), we canestimate that two key amino acids for protease inhibition include Met26(Kunitz domain 1) and Arg88 (Kunitz domain 2). Little is know about theinvolvement of different portions of UTI during inhibition of differentproteases, but removal of Kunitz domain 1 has been shown to changeproteases specificity, uncovering new inhibitory activity against FactorXa and plasma kallikrien. The full-length UTI does not show inhibitionof these two proteases (Morishita et al., Thrombosis Research 1994, vol73 (3/4) p 193-204). UTI also comprises two attached sugars, oneO-linked at Ser10 and one N-linked at Asn45. The half-life of UTI inrodents and humans is 4-30 minutes (Fries et al, International Journalof Biochemistry and Cell Biology, 2000, vol 32, p 125-137).

A UTI fusion protein should contain optimized sequence of amino acids,including the best start and stop points of any UTI domains, and may befused to another protein to enhance properties such as expression,purification, half-life, and stability. The exact sequence of the fusionpartner needs determination and may include variations in linkers,start/stop points, and/or mutations that may change the functionalproperties of the fusion partner.

Variants of ulinastatin obtained from urine are known WO199856916, U.S.Pat. Nos. 5,792,629, 5,407,915, 5,409,895, 7,019,123, and 6,583,108. Theconcept of fusion proteins of ulinastatin (and variations thereof) hasbeen disclosed US20080181892, U.S. Pat. No. 5,541,288, andUS20080255025. Certain UTI fusion proteins are described in CN103044554A. The fusion proteins of CN 103044554A relate to specificvariants in the Fc domain, presumably to avoid any Fc mediatedpharmacological effects (ADCC, CDC). We have surprisingly found that aUTI-Fc with wild type IgG1 is well tolerated and provides significantincrease in half-life. Also, compared to the UTI fusion proteins of CN103044554A the present UTI fusion proteins, in particular SEQ ID NO:1,demonstrate greater thermal stability.

The present invention provides UTI fusion proteins, pharmaceuticalcompositions comprising the same, preparation methods, and uses thereof.

SUMMARY OF THE INVENTION

The present invention provides UTI fusion proteins, comprising a UTIdomain and fusion partner wherein the UTI domain is operatively linkedto the fusion partner. The present invention provides UTI fusionproteins, comprising a UTI domain and an Fc domain wherein the UTIdomain is operatively linked to the Fc domain. The present inventionalso provides isolated UTI fusion proteins as described herein.

In some embodiments the present invention provides a UTI fusion protein,comprising a sequence comprising SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, and 29. In one embodiment, the present inventionprovides a UTI fusion protein comprising SEQ ID NO:1. In one embodiment,the present invention provides a UTI fusion protein comprising SEQ IDNO:3. In one embodiment, the present invention provides a UTI fusionprotein comprising SEQ ID NO:5. In one embodiment, the present inventionprovides a UTI fusion protein comprising SEQ ID NO:7. In one embodiment,the present invention provides a UTI fusion protein comprising SEQ IDNO:9. In one embodiment, the present invention provides a UTI fusionprotein comprising SEQ ID NO:11. In one embodiment, the presentinvention provides a UTI fusion protein comprising SEQ ID NO:13. In oneembodiment, the present invention provides a UTI fusion proteincomprising SEQ ID NO:15. In one embodiment, the present inventionprovides a UTI fusion protein comprising SEQ ID NO:17. In oneembodiment, the present invention provides a UTI fusion proteincomprising SEQ ID NO:19. In one embodiment, the present inventionprovides a UTI fusion protein comprising SEQ ID NO:21. In oneembodiment, the present invention provides a UTI fusion proteincomprising SEQ ID NO:23. In one embodiment, the present inventionprovides a UTI fusion protein comprising SEQ ID NO:25. In oneembodiment, the present invention provides a UTI fusion proteincomprising SEQ ID NO:27. In one embodiment, the present inventionprovides a UTI fusion protein comprising SEQ ID NO:29.

According to another embodiment of the present invention, the presentinvention provides a nucleic acid sequence encoding the UTI fusionproteins comprising the UTI fusion proteins described herein. Further,the invention provides the DNA sequences set forth as SEQ ID NO:2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30. In one embodiment,the nucleic acid encoding a UTI fusion protein further comprises avector containing control sequences to which the nucleic acid isoperably linked. In another embodiment, the present invention provides ahost cell comprising a nucleic acid sequence that encodes the UTI fusionprotein, such as a mammalian, insect, E. coli or yeast cell, andmaintaining the host cell under conditions in which the fusion proteinmolecule is expressed.

In another embodiment, the present invention provides a pharmaceuticalcomposition comprising the UTI fusion proteins described herein and apharmaceutically acceptable carrier or excipient.

According to a further embodiment of the present invention, there isprovided a method of treating UTI-related disorders, comprisingadministering to a patient in need thereof an effective amount of a UTIfusion protein described herein.

That is, the present invention provides for the use of a UTI fusionprotein as a medicament, including the manufacture of a medicament, andthe use of a UTI fusion protein described herein for the treatment ofthe UTI-related disorders described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 UTI domain structure and glycosylation sites.

FIG. 2 Two UTI-Fc constructs demonstrating altered linkers.

FIG. 3 Various UTI-Fc constructs of the present invention.

FIG. 4 DNA assemble strategy (SLIC) used in UTI fusion construction.

FIG. 5 Supression of protease activity (trypsin) by UTI and UTI-Fc1, SEQID NO:1

FIG. 6 Supression of protease activity (chymotrypsin) by UTI-Fc1, UFC1,SEQ ID NO:1.

FIG. 7 Supression of protease activity (multiple proteases) by UTI-Fc1,UFC1, SEQ ID NO:1.

FIG. 8 Supression of cytokine secreation (IL-6) by UTI and UTI-Fc1,UTI-Fc, SEQ ID NO:1.

FIG. 9 Purification yields of UTI fusion proteins.

FIG. 10 Effect of SEQ ID NO:1 on LPS Induced C5a in C3H Mice.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides UTI fusion proteins, a UTI domain andfusion partner wherein the UTI domain is operatively linked to thefusion partner. The UTI fusion proteins of the present invention have aninhibitory effect on proteases, including trypsin.

In some embodiments the fusion partner is an Fc polypeptide. In someembodiments the fusion partner is a human Fc polypeptide. In someembodiments the fusion partner is analog(s) of human Fc polypeptide. Insome embodiments the fusion partner is fragment(s) of human Fcpolypeptide. In other embodiments the the fusion partner is a mouse Fcpolypeptide. In other embodiments the the fusion partner is a rat Fcpolypeptide.

In some embodiments the fusion partner is human albumin. In someembodiments the fusion partner is an analog of human albumin. In someembodiments the fusion partner is a modified human albumin. In someembodiments the fusion partner is a fragment of human albumin.

In some embodiments the UTI domain is human UTI (hUTI). In someembodiments the UTI domain is an analog of hUTI. In some embodiments theUTI domain is a fragment of hUTI. In some embodiments, the UTI fusionprotein comprises a wild-type hUTI domain.

In some embodiments, the UTI fusion protein comprises a wild-typehumanUTI domain and a human Fc domain. In some embodiments, the UTIfusion protein comprises a wild-type hUTI domain, and a linker domain,and a human Fc domain.

In some embodiments, the UTI fusion protein comprises a wild-typehumanUTI domain and a human albumin or analog thereof or fragmentthereof. In some embodiments, the UTI fusion protein comprises wild-typehUTI domain, a linker domain, and a human albumin or analog thereof orfragment thereof.

In some embodiments, the Fc domain binds to an Fc receptor on a humancell. In some embodiments, the serum half-life of the molecule issignificantly longer than the serum half-life of the UTI domain alone.In some embodiments, the protease inhibitory activity of the UTI domainof the molecule is the same or greater than the UTI domain alone. Insome embodiments, administration of the molecule to a mouse decreasesinflammatory reactions, including, but not limited to, decreasing theactivation of immune cells or decreasing the production, secretion oractivity of cytokines or chemokines.

It is understood that the UTI domain may be operatively linked to thefusion partner by a linker domain.

The present invention provides a UTI fusion protein, comprising a UTIdomain fused to a polypeptide selected from the group consisting of a)Fc domain, b) an analog of the Fc domain, and c) fragment of the Fcdomain wherein the UTI domain is fused to the Fc domain, analog thereof,or fragment thereof by a linker domain. The present invention provides aUTI fusion protein, comprising a hUTI domain fused to a polypeptideselected from the group consisting of a) Fc domain, b) an analog of theFc domain, and c) fragment of the Fc domain wherein the hUTI domain isfused to the Fc domain, analog thereof, or fragment thereof by a linkerdomain.

The present fusion proteins encompass proteins having monomeric andmultimeric forms whether prepared by a digest of an intact antibody orproduced by other means.

The terms ‘multimer” and “multimeric” refers to proteins in which Fcdomains or molecules comprising Fc domains have two or more polypeptidechains associated covalently, non-covalently, or having both covalentand non-covalent interactions. The term multimer includes the termdimer.

The term “dimer” refers to proteins in which Fc domains or moleculescomprising Fc domains have two polypeptide chains associated covalently,non-covalently, or having both covalent and non-covalent interactions.That is, the term “dimer” refers to UTI fusion proteins in which two Fcdomains are associated covalently, non-covalently, or having bothcovalent and non-covalent interactions. More specifically, the term“dimer” refers to UTI fusion proteins in which two Fc domains areassociated covalently.

The present invention provides a UTI fusion protein, comprising a UTIdomain fused to a polypeptide selected from the group consisting of a)albumin, b) albumin analogs, c) fragments of albumin. The presentinvention also provides a UTI fusion protein, comprising a hUTI domainis fused to a polypeptide selected from the group consisting of a) humanalbumin, b) albumin analogs, c) fragments of human albumin wherein thehUTI is fused to the albumin, analog thereof, or fragment thereof by alinker domain.

Definition of Terms

The terms used in this specification and claims are defined as set forthbelow unless otherwise indicated.

As used herein, the terms “linked,” “fused,” or “fusion” are usedinterchangeably.

These terms refer to the joining together of two more elements orcomponents or domains, by whatever means including chemical conjugationor recombinant means. Methods of chemical conjugation are known in theart.

A “fusion protein” refers to a polypeptide having two or more portionscovalently linked together, where one or more of the portions is derivedfrom different proteins. The two portions may be linked directly by asingle peptide bond (e.g., the portions linked directly to each other)or through a peptide linker containing one or more amino acid residues(e.g. with an intervening amino acid or amino acid sequence between theportions). Generally, DNA encoding the two portions and the linker willbe in reading frame with each other and are produced using recombinanttechniques.

A “UTI domain” is a protein or peptide that mimics the activity of UTI.It is understood that the UTI domain of the present invention may bealtered such that they vary sequences from the naturally occurring ornative sequences from which they were derived, while retaining thedesired activity of the native sequence. Preferable the UTI domain isnative humanUTI (hUTI), analogs, and variants thereof. Variants of hUTIinclude replacing or modifying one or more amino acids of native hUTIthat are not a required structural feature or provide functionalactivity, including conservative substitutions. Variants of hUTI includeremoving or inserting one or more amino acids in native hUTI that arenot a required structural feature or provide functional activity.Variants of hUTI include replacing or modifying one or more amino acidsof native hUTI to modify one or more properties or activities. Variantsof hUTI include removing or inserting one or more amino acids in nativehUTI to modify one or more UTI properties or activities. Variants ofhUTI include removing or altering glycolsylation sites in nativehumanUTI. Variants of hUTI include removing or altering one or moreKunitz domain. Variants of hUTI can be introduced by standardtechniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis.

The amino acid residue sequence of the recombinant hUTI domain is setforth as SEQ ID NO:31. Generally, the UTI domain includes a sequence atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the recombinant hUTIdomain is set forth as SEQ ID NO:31.

An “Fc domain” is the polypeptide comprising the constant region of anantibody excluding the first constant region immunoglobulin domain andin some cases, part or all of the hinge. Thus, an Fc domain refers tothe non-antigen binding portion of an antibody, whether in monomeric ormultimeric form. The antibody from which the Fc domain arises ispreferably of human origin and may be any of the immunogobulins,although IgG1 and IgG2 are preferred.

An Fc domain includes the hinge region of the heavy chain. By “hinge” or“hinge region” or “antibody hinge region” or “immunoglobulin hingeregion” herein is meant the flexible polypeptide comprising the aminoacids between the first and second constant domains of an antibody, justupstream of the papian cleavage. Accordingly, for IgG, an Fc domaincomprises immunoglobulin domains CH2 and CH3 and the hinge regionbetween CH1 and CH2. Although the boundaries of the Fc region may vary,the human IgG heavy chain Fc region is usually defined to includeresidues C226 or P230 to its carboxyl-terminus, wherein the numbering isaccording to the EU index and in Kabat. In some embodiments, as is morefully described below, amino acid modifications are made to the Fcdomain, for example to alter binding to one or more FcγR receptors or tothe FcRn receptor.

Accordingly, in certain embodiments, the term Fc domain includes thehinge region which may be truncated, modified by replacement, deletionand/or insertion and further the modified or unmodified hinge region maybe the site of attachment of a linker domain.

An “analog of an Fc domain” refers to a molecule or sequence that ismodified from the native Fc but still comprises a binding site for thesalvage receptor. The term analog of an Fc domain includes a molecule orsequence that is humanized from a non-human native Fc. The term analogof an Fc domain also includes a molecule or sequence that lacks, or hasmodifications of, one or more native Fc residues that affect or areinvolved in disulfide formation, incompatibility with a host cell,N-terminal heterogeneity upon expression, stability, glycolsylation,interaction with a complement, binding to an Fc salvage receptor and/orinteraction with an Fcγ receptor.

The terms “fragments of the Fc domain” or “fragment of the Fc domain”refers to a native Fc from which one or more sites have been removedwhere the removed site(s) does not constitute structural features orfunctional activity that is required by the fusion proteins of thepresent invention. Fragments of the Fc domain include deleting residuesfrom the native Fc or truncating the native Fc and may includesubstitutions of the remaining residues. The inserted or alteredresidues (e.g., the substituted residues) may be natural amino acids oraltered amino acids, peptidomimetics, unnatural amino acids, or D-aminoacids.

Generally, the Fc domain includes a sequence at least 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, orIgM, in particular human IgG1 or IgG2.

The term Fc domain encompasses native Fc and analogs of Fc and includesmonomeric and multimeric forms whether prepared by a digest of an intactantibody or produced by other means.

In certain embodiments, an Fc domain comprises at least a hinge domain(upper, middle, and/or lower hinge region), a CH2 domain (or a variantor fragment thereof), and a CH3 domain (or a variant or fragmentthereof). In another embodiment, an Fc domain consists of a hinge domain(upper, middle, and/or lower hinge region), a CH2 domain (or a variantor fragment thereof), and a CH3 domain (or a variant or fragmentthereof). In certain other embodiments, an Fc domain consists of a hingedomain (upper, middle, and/or lower hinge region), a CH2 domain (or avariant or fragment thereof), a CH3 domain (or a variant or fragmentthereof), and a CH4 domain (or a variant or fragment thereof). Inanother embodiment, an Fc domain consists of a hinge domain (upper,middle, and/or lower hinge region) and a CH2 domain. In anotherembodiment, an Fc domain consists of a hinge domain (upper, middle,and/or lower hinge region) and a CH3 domain (or a variant or fragmentthereof). In another embodiment, an Fc domain consists of a CH2 domain(or a variant or fragment thereof), and a CH3 domain (or a variant orfragment thereof). In another embodiment, an Fc domain consists of acomplete CH2 domain and a complete CH3 domain. In another embodiment, anFc domain consists of a complete CH2 domain and a complete CH3 domain.In one embodiment, an Fc domain of the invention comprises at least theportion of an Fc molecule known in the art to be required for FcRnbinding. In another embodiment, an Fc domain of the invention comprisesat least the portion of an Fc molecule known in the art to be requiredfor Protein A binding. In another embodiment, an Fc domain of theinvention comprises at least the portion of an Fc molecule known in theart to be required for Protein G binding.

According to the present invention, an Fc domain generally refers to apolypeptide comprising all or part of the Fc domain of an immunoglobulinheavy-chain. As discussed above, this includes, but is not limited topolypeptides comprising the entire hinge region, CH1, CH2, and/or CH3domains as well as fragments of such peptides comprising, for example,the hinge, CH2 and CH3 domains. The Fc domain may be derived from anyimmunoglobulin of any species and/or subtype, including but not limitedto, a human IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody. AnFc domain includes the last two constant region immunoglobulin domainsof IgA, IgD, and IgG, the last three constant region immunoglobulindomains of IgE and IgM, and the flexible hinge N-terminal to thesedomains. For IgA and IgM, Fc may include the J chain.

An Fc domain as used herein encompasses native Fc and Fc variantmolecules. As with the Fc variants and native Fc proteins, the term Fcdomain includes molecules in monomeric and multimeric form, whetherdigested from an antibody or produced by other means.

As set forth herein it is understood, that any Fc domain may be modifiedsuch that it varies in amino acid sequence from the native Fc domain ofa naturally occurring immunoglobulin molecule. In certain exemplaryembodiments, the Fc domain retains an effector function, for example,FcγR binding. In certain exemplary embodiments, the Fc domain lacks aneffector function, for example, FcγR binding.

The Fc domain of the invention may be derived from differentimmunoglobulin molecules. For example, an Fc domain may comprise a CH2and/or CH3 domain derived from IgG1 and hinge region derived from IgG3.

In some embodiments, UTI fusion proteins include an Fc domain. Fcdomains useful for producing the UTI fusion proteins of the presentinvention may be obtained from a number of different sources. Inpreferred embodiments, an Fc domain of the UTI fusion proteins isderived from a human immunoglobulin. It is understood, however, that theFc domain may be derived from an immunoglobulin of another mammalianspecies, including for example, a rodent (e.g. a mouse, rat, rabbit,guinea pig) or non-human primate (e.g. Chimpanzee, Macaque) species.Moreover, the UTI fusion proteins Fc domain or portion thereof may bederived from any immunoglobulin class.

The terms “wild-type” or “wt” or “native” as used herein is meant anamino acid sequence or a nucleotide sequence that is found in nature,including allelic variations. A wild-type protein, polypeptide,antibody, immunoglobulin, IgG, polynucleotide, DNA, RNA, and the likehas an amino acid sequence or a nucleotide sequence that has not beenintentionally modified.

In certain embodiments the UTI fusion proteins of the present inventioncan employ a linker domain. The linker domain is used to operationallyconnect a UTI domain to a fusion partner.

The term “linker domain” refers to polypeptide linkers, non-peptidelinkers and combinations thereof. In particular, a linker domain can bea polypeptide. As used herein, the term “linker domain” refers to asequence which connects two domains in a linear sequence. As usedherein, the term “polypeptide linker” refers to a peptide or polypeptidesequence (e.g., a synthetic peptide or polypeptide sequence) whichconnects two domains in a linear amino acid sequence of a polypeptidechain. For example, polypeptide linkers may be used to connect a UTIdomain to an Fc domain. Preferably, such polypeptide linkers can provideflexibility to the polypeptide molecule. A UTI fusion protein of theinvention may comprise a linker domain, including a peptide linker.

For example, a linker domain can be used to connect two domains in alinear amino acid sequence of a polypeptide linker, such as linking aUTI domain with an Fc domain. In certain embodiments a linker domain canbe used to connect a UTI domain to a Fc domain. The linker domain can beused to connect the domains in any order. For example, in someembodiments a linker will connect a UTI domain and an Fc domain with theorder UTI-linker-Fc, whereas in other embodiments a linker will connecta UTI domain and an Fc domain with the order Fc-linker-UTI, where thepolypeptide regions are denoted from N-terminus to C-terminus. Exemplarypolypeptide linkers include those that consist of glycine and serineresidues, the so-called Gly-Ser polypeptide linkers. As used herein, theterm “Gly-Ser polypeptide linker” refers to a peptide that consists ofglycine and serine residues. An exemplary Gly-Ser polypeptide linkercomprises the amino acid sequence Ser(Gly₄Ser)_(n) wherein n is aninteger 1 to 10, SEQ ID NO:33-42, respectively. In one embodiment, theUTI fusion protein includes one or two Gly-Ser polypeptide linker(s) inwhich n=1. In one embodiment, the UTI fusion protein includes one or twoGly-Ser polypeptide linker(s) in which n=2. In one embodiment, the UTIfusion protein includes one or two Gly-Ser polypeptide linker(s) inwhich n=3. In one embodiment, the UTI fusion protein includes one or twoGly-Ser polypeptide linker(s) in which n=4. In one embodiment, the UTIfusion protein includes one or two Gly-Ser polypeptide linker(s) inwhich n=5. In one embodiment, the UTI fusion protein includes one or twoGly-Ser polypeptide linker(s) in which n=6. In one embodiment, the UTIfusion protein includes one or two Gly-Ser polypeptide linker(s) inwhich n=7. In one embodiment, the UTI fusion protein includes one or twoGly-Ser polypeptide linker(s) in which n=8. In one embodiment, the UTIfusion protein includes one or two Gly-Ser polypeptide linker(s) inwhich n=9. In one embodiment, the UTI fusion protein includes one or twoGly-Ser polypeptide linker(s) in which n=10.

Another exemplary linker is given in SEQ ID NO:43.

The term “comprising” means that a compound, i.e., fusion protein, mayinclude additional amino acids on either or both the N- or C-termini. Ofcourse, these additional amino acids should not significantly interferewith the activity of the compound, i.e., fusion protein.

The term “amino acid” refers to naturally occurring and synthetic aminoacids as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those encoded amino acids which are later modified, forexample, hydroxyproline and phosphoserine. Amino acid analogs refer tocompound, i.e., fusion proteins, that have the same basic chemicalstructure as the naturally occurring amino acids, that is, a carbon atombound to a hydrogen atom, carboxyl group, an amino group, and an Rgroup. Amino acid analogs have modified R groups, or give rise tomodified peptide backbones, but retain the same basic chemical structureas the naturally occurring amino acids.

The term “amino acid substitution” refers to the replacement of at leastone existing amino acid residue in a predetermined or native amino acidsequence with a different “replacement” amino acid.

The term “amino acid insertion” refers to the insertion of one or moreadditional amino acids into a predetermined or native amino acidsequence. The insertion can be one, two, three, four, five, or up totwenty amino acid residues.

The term “amino acid deletion” refers to removal of at least one aminoacid from a predetermined or native amino acid sequence. The deletioncan be one, two, three, four, five, or up to twenty amino acid residues.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein and refer to a polymer of amino acid residues.The terms apply to amino acid polymers in which one or more amino acidis a non-natural amino acid, synthetic amino acid, or amino acidmimetic.

The term “nucleic acid” refers to a deoxyribonucleotide orribonucleotide and polymers thereof in either single strand or doublestrand form. The term “nucleic acid” is used interchangeably with gene,nucleotide, polynucleotide, cDNA, DNA, and mRNA. Unless specificallylimited the term encompasses nucleic acids containing known analogues ofnatural nucleotides that have similar binding propertied as the naturalnucleic acid. Unless specifically limited, a particular nucleotidesequence also encompasses conservatively modified variants thereof (forexample, those containing degenerate codon substitutions) andcomplementary sequences as well as the as well as the sequencesspecifically described.

The polynucleotides of the present invention can be composed of anypolyribonucleotide or polydeoxyribonucleotide, which can be unmodifiedRNA or DNA or modified RNA or DNA. For example, polynucleotides can becomposed of single or double stranded regions, mixed single or doublestranded regions. In addition, the polynucleotides can be triplestranded regions containing RNA or DNA or both RNA and DNA. Modifiedpolynucleotides include modified bases, such as tritylated bases orunusual bases such as inosine. A variety of modification can be made toRNA and DNA, thus polynucleotide includes chemically, enzymactically, ormetabolically modified forms.

The term “derivatized” or derivative” refers to compound, i.e., fusionproteins, that have a cyclic portion, for example, cross-linked betweencysteinyl residues, the compound, i.e., fusion protein, is cross-linked,one or more peptidyl linkages is replaced by a non-peptide linkage, orthe N-terminus is replaced by a NRR₁, NRC(O)R₁, NRC(O)OR₁, NHC(O)NHR₁,NRS(O)₂R₂, succinamide or other group wherein R and R₁ are definedherein and/or the C-terminus is replaced with C(O)R₃ or NR₄R₅, andcompound, i.e., fusion proteins, in which amino acid moieties aremodified by treatment with agents capable of reacting with selected sidechains or terminal residues. R is selected from the group consisting ofhydrogen and C₁₋₆ alkyl, R₁ is selected from the group consisting ofhydrogen and C₁₋₆ alkyl, R₂ is selected from the group consisting ofC₁₋₆ alkyl, C₃₋₈ cycloalkyl, and optionally substituted phenyl; R₃ isselected from the group consisting of hydrogen, C₁₋₆ alkyl, and C₃₋₈cycloalkyl; R₄ is selected from the group consisting of hydrogen andC₁₋₆ alkyl; R₅ is selected from the group consisting of hydrogen, C₁₋₆alkyl and C₃₋₈ cycloalkyl; or R₄ and R₅ are taken together with thenitrogen to which they are attached form a 4 to 7 membered, saturated,ring optionally having 1 additional ring heteroatom selected from thegroup N, O, and S.

The term “C₁₋₆ alkyl” refers to a straight or branched alkyl chain ofone to six carbon atoms.

The term “C₃₋₈ cycloalkyl” refers to monocyclic or bicyclic, saturatedor partially (but not fully) unsaturated alkyl ring of three to eightcarbon atoms, and includes cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, and the like. It is understood that the term includesbenzofused cyclopentyl and cyclohexyl.

The term “optionally substituted phenyl” refers to a phenyl groupoptionally substituted with 1 to 3 substituents independently selectedfrom the group consisting of halo, C₁₋₆ alkyl, C₁₋₆ alkoxy, cyano, andtrifluoromethyl.

Preparation

The compounds, i.e., fusion proteins, of this invention may be preparedby standard synthetic methods, recombinant DNA techniques, or othermethods of preparing peptides and fusion proteins. In an exemplaryprocess a hUTI domain is covalently linked to an Fc domain by expressionof a DNA construct encoding the UTI domain and the Fc domain and anylinker domain.

Alternative ways to construct a UTI fusion protein are envisioned. Insome embodiments, the domain orientation can be altered to construct anFc-UTI molecule or a UTI-Fc molecule or a UTI-Fc-UTI molecule thatretains FcR binding and has active UTI domain.

In some embodiments, UTI fusion proteins include a wild-type Fc domainthat can allow the fusion protein to undergo endocytosis after bindingFcRn (Fc neonatal receptor). Thus, the present invention furtherprovides methods for producing the disclosed UTI fusion proteins. Thesemethods encompass culturing a host cell containing isolated nucleicacid(s) encoding the UTI fusion proteins of the invention. As will beappreciated by those in the art, this can be done in a variety of ways,depending on the nature of the UTI fusion protein. In some embodiments,the UTI fusion protein of the invention is produced and can be isolated.

In general, nucleic acids are provided that encode for the UTI fusionprotein of the invention. Such polynucleotides encode for a UTI domain,the fusion partner, and any linker domain. The present invention alsocontemplates oligonucleotide fragments derived from the disclosedpolynucleotides and nucleic acid sequences complementary to thesepolynucleotides.

The polynucleotides can be in the form of RNA or DNA. Polynucleotides inthe form of DNA, cDNA, genomic DNA, nucleic acid analogs, and syntheticDNA are within the scope of the present invention. The DNA may bedouble-stranded or single-stranded, and if single stranded, may be thecoding (sense) strand or non-coding (anti-sense) strand. The codingsequence that encodes the polypeptide may be identical to the codingsequence provided herein or may be a different coding sequence, whichsequence, as a result of the redundancy or degeneracy of the geneticcode, encodes the same polypeptides as the DNA provided herein.

In some embodiments, nucleic acid(s) encoding the UTI fusion proteins ofthe invention are incorporated into expression vectors, which can beextrachromosomal or designed to integrate into the genome of the hostcell into which it is introduced. Expression vectors can contain anynumber of appropriate regulatory sequences (including, but not limitedto, transcriptional and translational control sequences, promoters,ribosomal binding sites, enhancers, origins of replication, etc.) orother components (selection genes, etc.), all of which are operablylinked as is well known in the art. In some cases two nucleic acids areused and each put into a different expression vector (e.g. heavy chainin a first expression vector, light chain in a second expressionvector), or alternatively they can be put in the same expression vector.It will be appreciated by those skilled in the art that the design ofthe expression vector(s), including the selection of regulatorysequences may depend on such factors as the choice of the host cell, thelevel of expression of protein desired, etc.

In general, the nucleic acids and/or expression can be introduced into asuitable host cell to create a recombinant host cell using any methodappropriate to the host cell selected (e.g., transformation,transfection, electroporation, infection), such that the nucleic acidmolecule(s) are operably linked to one or more expression controlelements (e.g., in a vector, in a construct created by processes in thecell, integrated into the host cell genome). The resulting recombinanthost cell can be maintained under conditions suitable for expression(e.g. in the presence of an inducer, in a suitable non-human animal, insuitable culture media supplemented with appropriate salts, growthfactors, antibiotics, nutritional supplements, etc.), whereby theencoded polypeptide(s) are produced. In some cases, the heavy chains areproduced in one cell and the light chain in another.

Mammalian cell lines available as hosts for expression are known in theart and include many immortalized cell lines available from the AmericanType Culture Collection (ATCC), Manassas, Va. including but not limitedto Chinese hamster ovary (CHO) cells, HEK 293 cells, NSO cells, HeLacells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), humanhepatocellular carcinoma cells (e.g., Hep G2), and a number of othercell lines. Non-mammalian cells including but not limited to bacterial,yeast, insect, and plants can also be used to express recombinantantibodies. In some embodiments, the antibodies can be produced intransgenic animals such as cows or chickens.

In an embodiment, the fusion proteins of the invention are encoded by anucleotide sequence. Nucleotide sequences of the invention can be usefulfor a number of applications, including: cloning, gene therapy, proteinexpression and purification, mutation introduction, DNA vaccination of ahost in need thereof, antibody generation for, e.g., passiveimmunization, PCR, primer and probe generation, siRNA design andgeneration and the like. In an embodiment, the nucleotide sequence ofthe invention comprises, consists of, or consists essentially of, anucleotide sequence selected from SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, or 32.

In an embodiment, a nucleotide sequence includes a nucleotide sequenceat least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotidesequence set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, or 32. In an embodiment, a nucleotide sequence includesa contiguous nucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to a contiguous nucleotide sequence set forth in SEQ ID NO:2,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32.

Preferred UTI fusion proteins of the invention comprise a sequence(e.g., at least one Fc domain) derived from a human immunoglobulinsequence. However, sequences may comprise one or more sequences fromanother mammalian species. For example, a primate Fc domain or nucleasedomain may be included in the subject sequence. Alternatively, one ormore murine amino acids may be present in a polypeptide. In someembodiments, polypeptide sequences of the invention are not immunogenicand/or have reduced immunogenicity. The UTI fusion proteins of theinvention may comprise conservative amino acid substitutions at one ormore amino acid residues, e.g., at essential or non-essential amino acidresidues. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art, including basic side chains (e.g.,lysine, arginine, histidine), acidic side chains (e.g., aspartic acid,glutamic acid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a nonessential amino acidresidue in a binding polypeptide is preferably replaced with anotheramino acid residue from the same side chain family. In anotherembodiment, a string of amino acids can be replaced with a structurallysimilar string that differs in order and/or composition of side chainfamily members. Alternatively, in another embodiment, mutations may beintroduced randomly along all or part of a coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be incorporatedinto binding polypeptides of the invention and screened for theirability to bind to the desired target.

Uses

In one embodiment, the invention provides methods of diagnosing andtreating UTI-related conditions. As used herein terms “condition,”“disorder,” and “disease” relate to any unhealthy or abnormal state. Theterm “UTI-related conditions” includes conditions, disorders, anddiseases in which UTI provides a therapeutic benefit. The term“UTI-related conditions” include conditions characterized by animmunomodulatory or an inflammatory effect. In particular, the termUTI-related conditions include pancreatitis, including acutepancreatitis and chronic pancreatitis, systemic inflammatory responsesyndrome, acute circulatory failure (e.g., caused by shock),disseminated intravascular coagulation, and multiple organ dysfunctionsyndrome. The term UTI-related conditions also include use in high-risksurgical patients. The term UTI-related conditions also includesinfections of the lung, liver, heart, or kidney. The term UTI-relatedconditions also includes severe sepsis. The term UTI-related conditionsalso includes acute lung injury (ALI) caused by SARS viruses or acuterespiratory distress syndrome (ARDS).

In one embodiment, the invention provides methods of treating aUTI-related condition, comprising administering to a patient in needthereof an effective amount, e.g., a pharmaceutically effective amount,of a disclosed UTI fusion protein. In certain embodiments, the conditionis one specifically mentioned herein.

The pharmaceutical compositions of the present invention are prepared ina manner well known in the pharmaceutical art and include at least oneof UTI fusion protein of the invention as the active ingredient.Pharmaceutical composition of the UTI fusion proteins used in accordancewith the present invention are prepared by mixing a UTI fusion proteinhaving the desired degree of purity with optional pharmaceuticallyacceptable excipients. The term “pharmaceutically acceptable excipient”refers to those typically used in preparing pharmaceutical compositionsand should be pharmaceutically pure and non-toxic in the amounts used.They generally are a solid, semi-solid, or liquid material which in theaggregate can serve as a vehicle or medium for the active ingredient.Some examples of pharmaceutically acceptable excipients are found inRemington's Pharmaceutical Sciences and the Handbook of PharmaceuticalExcipients and include diluents, vehicles, carriers, sustained releasematrices, stabilizing agents, preservatives, solvents, suspendingagents, buffers, emulsifiers, dyes, propellants, coating agents, andothers. Generally for injection or intravenous administration the UTIfusion proteins of the present invention are in the form of lyophilizedformulations, or aqueous solutions.

Pharmaceutically acceptable excipients are nontoxic to subjects in theamounts used, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The pharmaceutical compositions herein may also contain more than oneactive compound, i.e., fusion protein, as necessary for the particularindication being treated, preferably those with complementary activitiesthat do not adversely affect each other. Such molecules are suitablypresent in combination in amounts that are effective for the purposeintended.

The pharmaceutical compositions to be used for in vivo administrationshould be sterile, or nearly so. This is readily accomplished byfiltration through sterile filtration membranes.

The UTI fusion proteins of the invention are administered to a subject,in accord with known methods, such as intravenous administration as abolus or by continuous infusion over a period of time, by intramuscular,intraperitoneal, intracerobrospinal, subcutaneous, intra-articular,intrasynovial, or intrathecal injection or infusion or by topical orinhalation routes. Intravenous or subcutaneous administration of the UTIfusion protein is preferred.

The terms “treat,” “treatment,” and “treating” include improvement ofthe conditions described herein. The terms “treat,” “treatment,” and“treating” include all processes providing slowing, interrupting,arresting, controlling, or stopping of the state or progression of theconditions described herein, but does not necessarily indicate a totalelimination of all symptoms or a cure of the condition. The terms“treat,” “treatment,” and “treating” are intended to include therapeutictreatment of such disorders. The terms “treat,” “treatment,” and“treating” are intended to include prophylactic treatment of suchdisorders.

As used herein the terms “patient” and “subject” includes humans andnon-human animals, for example, mammals, such as mice, rats, guineapigs, dogs, cats, rabbits, cows, horses, sheep, goats, and pigs. Theterm also includes birds, fish, reptiles, amphibians, and the like. Itis understood that a more particular patient is a human. Also, moreparticular patients and subjects are non-human mammals, such as mice,rats, and dogs.

As used herein, the term “effective amount” refers to the amount ofcompound, i.e., fusion protein, of the invention which treats, uponsingle or multiple dose administration, a patient suffering from thementioned condition. An effective amount can be readily determined bythe attending diagnostician as a medical professional, such as aphysician or a veterinarian as one skilled in the art, by the use ofknown techniques and by observing results obtained under analogouscircumstances. For example, a medical professional could start doses ofthe medicament employed in the pharmaceutical composition at levelslower than that required in order to achieve the desired therapeuticeffect and gradually increase the dosage until the desired effect isachieved.

In determining the effective amount, the dose, a number of factors areconsidered by the attending diagnostician, including, but not limitedto: the species of patient; its size, age, and general health; thespecific condition, disorder, or disease involved; the degree of orinvolvement or the severity of the condition, disorder, or disease, theresponse of the individual patient; the particular compound, i.e.,fusion protein, administered; the mode of administration; thebioavailability characteristics of the preparation administered; thedose regimen selected; the use of concomitant medication; and otherrelevant circumstances. Specific amounts can be determined by theskilled person. Although these dosages are based on an average humansubject having a mass of about 60 kg to about 70 kg, the physician willbe able to determine the appropriate dose for a patient (e.g., aninfant) where the mass falls outside of this weight range.

Dosage regimens are adjusted to provide the desired response. Forexample, a single bolus may be administered, several divided doses maybe administered over time or the dose may be proportionally reduced orincreased as indicated by the exigencies of the therapeutic situation.

Parenteral compositions may be formulated in dosage unit form for easeof administration and uniformity of dosage. Dosage unit form as usedherein refers to physically discrete units suited as unitary dosages forthe subjects to be treated; each unit contains a predetermined quantityof active compound, i.e., fusion protein, calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier.

The present pharmaceutical compositions are preferably formulated in aunit dose form, each dose typically containing from about 0.5 mg toabout 100 mg of a UTI fusion protein of the invention. The term “unitdose form” refers to a physically discrete unit containing apredetermined quantity of active ingredient, in association with asuitable pharmaceutical excipient, by which one or more is usedthroughout the dosing regimen to produce the desired therapeutic effect.One or more “unit dose form” may be taken to affect the treatmentdosage.

An exemplary, non-limiting range for an effective amount of a UTI fusionprotein used in the present invention is about 0.1-100 mg/kg, such asabout 0.1-50 mg/kg, for example about 0.1-20 mg/kg, such as about 0.1-10mg/kg, for instance about 0.5 mg/kg, about such as 0.3 mg/kg, about 1mg/kg, or about 3 mg/kg. In another embodiment, the UTI fusion proteinis administered in a dose of 1 mg/kg or more, such as a dose of from 1to 20 mg/kg, e.g. a dose of from 5 to 20 mg/kg, e.g. a dose of 8 mg/kg.An exemplary, non-limiting range for an effective amount of a UTI fusionprotein used in the present invention is about 1-500 mg/dosage, such asabout 1-100 mg/dosage, for example about 1-50 mg/dosage, such as about1-10 mg/dosage, for instance about 1 mg/dosage, or about 3 mg/dosage, orabout 5 mg/dosage.

In one embodiment, the UTI fusion protein is administered by infusion inan every 3 days or weekly dosage of from 10 to 500 mg/dosage. Suchadministration may be repeated as necessary to maintain the desiredtherapeutic effect.

As non-limiting examples, treatment according to the present inventionmay be provided a dosage of UTI fusion protein in an amount of about0.1-100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on atleast one of day; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, or 40 mg/kg, or alternatively, at least once a week1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20mg/kg after initiation of treatment, or any combination thereof. Asnon-limiting examples, treatment according to the present invention maybe provided a dosage of UTI fusion protein in an amount of about 1-100mg/dosage, such as 1, 5, 10, 20, 30, 40, 45, 50, 60, 70, 80, 90, 100150, 200, 250, 300, 350, or 400 mg/dosage. On at least once a day 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or40 mg/dosage after initiation of treatment, or any combination thereof.On at least one of week 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100mg/dosage after initiation of treatment, or any combination thereof.

The UTI fusion proteins of the invention find use in a variety ofapplications, including treatment of UTI-related diseases. UTI fusionproteins of the invention may find use in treating diseases with immunesystem involvement, autoimmune diseases, inflammatory diseases,post-operative inflammatory responses, lysosome-associated diseases,coagulation diseases protease-related diseases and as an adjuvanttherapy during surgery. UTI fusion proteins of the invention may finduse in treating pancreatitis (including endoscopy-induced pancreatitisand acute pancreatitis), arthritis, SARS, systemic inflammatory responsesyndrome, acute circulatory failure, sepsis, hepatitis, appendicitis,colitis, organ failure, organ damage (including pancreas, kidney, lung),reperfusion injury, Stevens-Johnson syndrome, toxic epidermalnecrolysis, shock, ischemic injuries, acute lung injury (including thatcaused by acute aortic dissection), asthma, lung inflammation, pneumonia(including ventilator-associated), disseminated intravascularcoagulation (DIC), acute respiratory distress syndrome (ARDS), andsystemic inflammatory response syndrome.

UTI fusion proteins of the present invention may find use in inhibitingproteases, including the serine proteases, including, trypsin,chymotrypsin, thrombin, kallikrein, plasmin, elastase, cathepsin,lipase, hyaluronidase, factors IXa, Xa, XIa, and XIIa, andpolymorphonuclear leukocyte elastase.

UTI fusion proteins of the present invention may find use in suppressionof proinflammatory mediators, such as cytokines, tumor necrosisfactor-alpha, interleukin-1, -1β, -4, -6 and -8, -10 and chemokines.

UTI fusion proteins of the present invention may find use in treatmentof cancer, including the prevention of tumor invasion and metastasis,altered rates of apoptosis, and reduction of loss of renal function incisplatinum treatment.

UTI fusion proteins of the present invention may find use to treat AIDS,including as adjunctive treatment.

EXAMPLES

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for. The practice of the presentinvention will employ, unless otherwise indicated, conventional methodsof protein chemistry, biochemistry, recombinant DNA techniques andpharmacology, within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., T. E. Creighton, Proteins: Structureand Molecular Properties (W.H. Freeman and Company, 1993); A. L.Lehninger, Biochemistry (Worth Publishers, Inc.); Sambrook, et al,Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods InEnzymology (S. Colowlck and N. Kaplan eds., Academic Press, Inc.);Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.: MackPublishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry3rd Ed. (Plenum Press) Vols A and B (1992).

Example 1: Construction of DNA Vectors Encoding UTI Fusion Proteins

Methods for performing molecular biology are known in the art and can befound, for example, in Molecular Cloning: A laboratory Manual 4thedition (Micheal Green and Joseph Sambrook, Cold Spring Harbor Press,2012).

A gene encoding UTI-Fc1 was ordered using the GeneArt codon-optimizedgene synthesis services from Life Technologies (Carlsbad, Calif.). Theprotein sequence is as listed in SEQ ID NO: 1 with a signal peptide,MGWSCIILFLVATATGVHS, added for secretion. FIG. 1 shows the generalregions of UTI used in the fusion. The gene encoding UTI-Fc1 was ligatedinto a mammalian expression vector. Mammalian expression vectors areknown in the art including pSecTag2/Hygro A, pcDNA4 and pcDNA6 vectors(Life Technologies, Carlsbad Calif.). The vector was digested with therestriction enzymes, HindIII-HF and EcoRI from New England Biolabs(NEB). This fragment was ligated into the expression vector whichprovides carbenicillin resistance and had been digested with the sametwo restriction enzymes. A vector: insert molar ratio of 1:3 was used inligation. Ligated DNA was transformed into 10-beta chemically competentE. coli cells from NEB, and plated on LB-Carbenicillin plates forovernight growth. Colonies were grown overnight in LB with Carbenicillinand miniprep DNA was prepared by Qiagen's QIAprep Spin Miniprep Kit(Qiagen, Hilden, Germany). DNA was then sequenced using DNA sequencingservices from Bio Applied Technologies Joint (BATJ, San Diego). Thesequence verified colony was subsequently grown in LB media withcarbenicillin for DNA purification with the BenchPro 2100 instrument andMaxiCard from Life Technologies.

Example 2: Construction of DNA Vectors Encoding Variants of UTI-FcFusion Proteins

SEQ ID NOS:1-28 list DNA and protein sequences of some UTI-Fc fusionproteins. These UTI fusion proteins comprise modifications that alter Igisotype, linkers, UTI domain, UTI and Fc domain order (N- orC-terminal), UTI species, Fc species, UTI start/stop residues, sugarattachment, protease sensitive sites, and Fc effector function. SomeUTI-Fc proteins are depicted in FIGS. 2 and 3. UTI-Fc fusion proteinscomprising three amino acid modifications to Ser (IgG1 Fc3Ser,C154S/P172S/P265S) comprises mutations to alter disulfide bond formationand FcγR functions.

Creation of UTI-Fc Expression Constructs

The nucleotide sequences of the UTI (e.g. wild-type, S10A, and K21S K22Svariants) and human Fc3Ser domains were codon optimized for CHO cellexpression and synthesized by Life Technologies (Carlsbad, Calif.). Thefollowing constructs were created in a CHO expression vector byassembling UTI and Fc3Ser domains via sequence and ligation-independentcloning (SLIC) method (Li and Elledge 2007 Nature Methods 4(3):251-256):UTI-Fc3Ser, UTI S10A-Fc3Ser, UTI K21S K22S-Fc3Ser, UTI m2-Fc3Ser, UTIL1-Fc3Ser, UTI L2-Fc3Ser (FIG. 4A). SLIC-based DNA assembly was done bymixing the linearized vector (30 ng), UTI (100 ng) and Fc3Ser (100 ng)PCR products with appropriate overhang sequences for homologousrecombination, and T4 DNA polymerase (0.5 U) in 5 μL volume containingNEBuffer 2 and BSA (New England Biolabs). After 30 min incubation atroom temperature, the exonuclease activity of T4 DNA polymerase wasquenched by adding 2 mM of dCTP. Then in vitro homologous recombinationwas done by temperature gradient from 75 C to 37 C over 30 min. Thereaction mixture containing assembled DNA was chemically transformedinto TOP10 E. coli (Invitrogen), and plated on LB-agar containingcarbenicillin. The open reading frames for the remaining constructs (UTIm1-Fc3Ser, UTI d1-Fc3Ser, UTI d2-Fc3Ser, UTI L3-Fc3Ser, UTI-Fc IgG2,Fc3Ser-UTI, and mouse UTI-mouse IgG1) were codon optimized andsynthesized as fusion constructs. These constructs were cloned into theexpression vector using SLIC method as described above (FIG. 4B). TheDNA sequences of all the 13 constructs in the vector were verified bySanger DNA sequencing.

Example 3: Expression of UTI-Fc Fusions in CHO Cells

A DNA vector encoding UTI-Fc1 was stably transfected into CHO—S cellsusing Invitrogen's Freestyle MAX Reagent. Bulk cultures were plated intoT-flasks and selected using CD CHO supplemented with variousconcentrations of methionine sulfoximine (MSX) ranging from 50-100 μM.Once the cultures recovered from selection, they were expanded forproduction and cryopreservation. Multiple production batches were doneto support in vitro and in vivo testing. The production process is a10-14 day fed-batch culture using CD FortiCHO, CD Efficient Feed B, andCD Efficient Feed C from Invitrogen. The production volumes ranged from1L-3L, and the cultures were harvested by centrifugation at 3500 rpm for1-2 hours followed by sterile filtration of the supernatant and theresulting cell supernatant was used in purification.

Example 4: Purification of UTI-Fc Fusion Proteins

Purification of 2 batches of UTI-Fc1 was done by applying 2.3 liters CHOcell conditioned media with expressed UTI-Fc1 to 30 ml protein A mAbselect 1× (GE healthcare) equilibrated in 25 mM trisodium citrate pH8.1, 125 mM NaCl. The column was washed with 2 column volumes (60 ml) 25mM trisodium citrate pH 8.1, 125 mM NaCl and then with 2 column volumes(60 ml) 25 mM trisodium citrate pH 8.1, 2000 mM NaCl. The column wasthen equilibrated with 2 column volumes (60 ml) 25 mM trisodium citratepH 8.1, 125 mM NaCl. UTI-Fc1 was eluted with a 7 column volume (210 ml)gradient to 100% 25 mM citric acid pH 2.9, 125 mM NaCl. The UTI-Fc1eluted as two peaks, a broad, flanking peak at an approximate pH of 5.5and a sharper peak at an approximate pH of 3.5. Then, the concentratedUTI-Fc was buffer exchanged into a final buffer of TBS pH 7.4 (25 mMtris, 130 mM NaCl, 2.7 mM KCl pH 7.4) using Amicon Ultra centrifugalconcentrators with a 30K M.W.C.O. Purified protein yields are shown inTABLE 1 and the protein was stored at −80° C. for further use.

TABLE 1 Final Final Protein % of Peak product product A Column totalvolume conc. Yield Yield protein Peak name (mL) (mg/mL) (mg) (%) load1st batch peak 40 10 110 59 52 2nd batch peak 40 11 150 72 68

Example 5: Expression and Purification of Additional UTI-Fc FusionProteins

On the day of transfection, CHO cells were counted and seeded at densityof 2.2×10{circumflex over ( )}6 live cells/mL in 90% of total volume—900mLs and grown in shaker flasks at 33° C. until transfection. Frozen DNAwas thawed and added to PEI (Polyethylenimine—a cationic polymer) andAKT. DNA was added at 0.625 ug/1 million cells. 1L of cells requires1.25 mg. 90% of the total DNA added is DNA of interest=1.125 mgs. Theremaining 10% was AKT (encodes anti-apoptotic protein)=0.125 mgs. PEIwas added at 2.5 ug/1 million cells. For a 1 L transfection this was 5mg. The PEI solution was added to the diluted DNA and incubated at roomtemperature for 15 minutes before addition of the DNA complex to thecells.

Cultures were grown at 33° C., 5% CO2, and 125 rpm. 1 to 4 hourspost-transfection, 0.6 mM Valproic acid was added. For the 1Ltransfection, this was 2 mL of 300 mM stock. On day 1, 1:250anti-clumping agent was added i.e. 4 mLs/1L and 15% v/v CD EfficientFeed C i.e. 150 mLs/1L. On Day 5 and Day 9 15% CD Efficient Feed C wasadded.

Cells supernatents were harvested on Day 14, cells were counted andprotein titers were determined. Cells were spun down by centrifugationat 3000 rpm for 30 minutes at 4° C. The supernatants were filteredthrough a 0.2 micron filter and stored at 4° C. or frozen at −20° C.

Purification of UTI-Fc fusions was done by Protein A chromatography. 200mL of cell culture supernant was mixed with 2 ml of MabSelect SureProtein A Sepharose beads and shaken overnight at 4° C. The bead mixturewas then centrifuged in 50 ml tubes at 1200 rpm for 5 minutes and thesupernatant was discarded. The beads are added to a column and washedthrice with binding buffer (Biorad, Hercules, Calif.). UTI-fusions wereeluted with 8 ml MAPS II Elution buffer (Biorad, Hercules, Calif.). 2 mlof neutralization solution (1M Tris-HCl pH 8) was added. Samples werethen buffer exchanged into 25 mM Citrate, 125 mM NaCl, pH 5.5 byrepeated concentration and dilution with the buffer using AmiconCentrifugal units (30 MWCO, 15 mLs, Millipore). FIG. 9 lists thepurifications results of various UTI fusion proteins.

Example 6: Inhibition of Proteases by UTI Fusion Proteins

In Vitro Enzymatic Assay for Trypsin Inhibition by UTI-Fc Fusion Protein

Solutions of UTI-Fc1 at various concentrations (≤200 nM finalconcentration) are prepared in 50 mM HEPES, 150 mM NaCl, 20 mM CaCl2 and0.01% Brij L23, pH 7.4. Activity assays were performed in Greiner384-well small volume plates. All steps were conducted at ambienttemperature.

Human pancreatic trypsin (1.5 nM final concentration) (Athens Research &Technology, Inc) was added to the dilutions then pre-incubated with thetest UTI-Fc for 15 minutes. Next, the reaction was initiated with 100 μM(final) of substrate N□-Benzoyl-L-arginine-7-amido-4-methylcoumarinhydrochloride SIGMA B7260-25MG. The reaction mixture total volume was 20□l. Trypsin activity was determined via fluorescence. For example, thefluorescence intensity was determined in kinetic mode over a window of30 to 60 minutes on a BMG PHERAstar FS or PHERAstar plus using anexcitation wavelength of 370 nm and an emission wavelength of 470 nm.Trypsin activity was linearly proportional to the change in fluorescenceobserved (final-initial). The percent inhibition of Trypsin at a givenUTI-Fc concentration was defined as:

Percent inhibition=100*(1−((Fi−Fp)/(Fn−Fp)))

Where: Fi was the observed fluorescence at a given concentration of testUTI-Fc.

Fp was the observed fluorescence of a positive control i.e., the averagevalue of 2 to 6 assays in the absence of Trypsin.

Fn was the observed fluorescence of a negative control i.e., the averagevalue of 2 to 6 assays of Trypsin in the presence of vehicle alone.

The IC50 (the molar concentration of the compound, i.e., fusion protein,that produces 50% inhibition) of a test compound, i.e., fusion protein,was calculated by non-linear least squares curve fitting of the equationPercent inhibition=Bottom+((Top-Bottom)/(1+((IC50/[UTI-Fc]){circumflexover ( )}Hill))). Included within the panel of UTI-Fc was one positivecontrol. As shown in FIG. 5, humanUTI has an IC50 of ˜3 nM.

Measurement of the inhibition of other proteases by UTI-Fc1 was alsomeasured at Reaction Biology Corporation (Malvern, Pa.). FIG. 6demonstrates UTI-Fc1's inhibition of chymotrypsin. FIG. 7 lists theinhibitory constants of UTI-Fc1 for a variety of proteases. UTI-Fc1inhibits chymotrypsin and plasmin moderately and shows weak inhibitionof caspase-1, cathepsin C, and papain.

Example 7: Cellular Effects of Treatment with UTI Fusion Proteins

UTI-Fc1 inhibition of cytokine release was measure in a cell-basedassay. BEAS2B cells were seeded at the density of 20,000 cells/well in96 well plate and cultured using complete BEGM Bullet Kit (Lonza) in CO2incubator. After 24 hours, the culture media were replaced with plainDMEM for starvation and cells were cultured overnight. Then cells wereincubated with fresh plain DMEM containing 100 nM trypsin with variousconcentrations of human urine UTI or recombinant UTI-Fc1 proteins. After8 hours, culture supernatants were collected and IL-6 protein levelswere assessed using human IL-6 DuoSet (R&D Systems).

The results demonstrate that both UTI and UTI-Fc decreasedtrypsin-induced IL-6 production in BEAS2B cells. As shown in FIG. 8,inhibition was dose-dependent with the IC50 values of 0.40 and 0.41μg/mL, respectively.

Example 8

Stability Measurements of UTI-Fc Molecules—Thermal Denaturation

Assays were performed to measure the thermal and real-time stability.All molecules demonstrated activity in trypsin inhibition. Thermalstability was measured by differential scanning calorimetry on aMicrocal VP-DSC calorimeter. Samples were prepared at 1 mg/ml andbuffered in 0.25 mM Tris pH7.4, 0.13M NaCl and 0.0027M KCl. Samples wereheated from 25° C. to 110° C. at a rate of 200° C. per hour. UTI-Fc1 wascompared to Application Publication Number CN 103044554 A, SEQ ID's 2and 6, which comprise an IgG2 or IgG1 Fc domain respectively. Theresults are presented in Table 8.

TABLE 8 DSC Tm1 DSC Tm2 Protein (° C.) (° C.) UTI-Fc1 (SEQ ID NO: 1)70.86 85.79 CN 103044554 A SEQ ID 6 68.72 86.38 CN 103044554 A SEQ ID 268.47 79.22

Example 9

Real-Time Stability Measurements

Real-time stability measurements were performed by incubating SEQ IDNO:1 (UTI-Fc1) or CN 103044554 A, SEQ IDS 2 or 6 at 2-8° C., and 40° C.for 0, 2 and 4 weeks in TBS, pH7.4 buffer. The formation of higher andlower molecular weight species was determined by size exclusionchromatography (SEC) and visualized with polyacrilamide gelelectrophoresis (PAGE). The concentration of each UTI-Fc was alsomonitored by determination of the absorbance of the solution at 280 nm(A280), using extinction coefficients determine by the proteincomposition. The UTI-Fc molecules produce two, partially overlappingpeaks, when analyzed by SEC. The percentage peak area in each UTI-Fcsample measured by SEC is reported in Table 9 at time=0 weeks, 2 weeks,and 4 weeks. Also shown is the percentage change in concentrationmeasured by A280 (% Δ (mg/ml)) at time=2 weeks and 4 weeks. The initialTO concentration of each sample was UTI-Fc1=33.5 mg/mL, CN 103044554 ASEQ ID 2=8.5 mg/mL, and CN 103044554 A SEQ ID 6=5.6 mg/mL. A variabilityof 3% is typical for SEC and 15% for individual UV measurements.Analysis by PAGE showed that each UTI-Fc molecule showed the expectedbanding pattern for full-length UTI-Fc.

TABLE 9 % Δ % Δ SEC SEC SEC (mg/ml) (mg/ml) Protein 0 weeks 2 weeks 4weeks 2 weeks 4 weeks UTI-Fc1 Peak1 NA NA NA (SEQ ID NO: 1) 35.7% Peak264.3% UTI-Fc1 NA Peak1 Peak1 9.2% 1.2% (SEQ ID NO: 1) 36.2% 36.1% at2-8° C. Peak2 Peak2 63.8% 63.9% UTI-Fc1 NA Peak1 Peak1 0.2% 11.9% (SEQID NO: 1) 37.1% 36.8% at 40° C. Peak2 Peak2 62.9% 63.2% CN 103044554 APeak1 NA NA NA NA SEQ ID 2 30.6% Peak2 69.4% CN 103044554 A NA Peak1Peak1 0.2% 2.7% SEQ ID 2 29.0% 31.5% at 2-8° C. Peak2 Peak2 71.0% 68.5%CN 103044554 A NA Peak1 Peak1 1.6% 3.5% SEQ ID 2 28.2% 30.3% at 40° C.Peak2 Peak2 71.8% 69.7% CN 103044554 A Peak 1 NA NA NA NA SEQ ID 6 36.6%Peak 2 63.4% CN 103044554 A NA Peak1 Peak1 7.8% 9.5% SEQ ID 6 34.8%36.4% at 2-8° C. Peak2 Peak2 65.2% 63.6% CN 103044554 A NA Peak1 Peak13.6% 6.0% SEQ ID 6 33.9% 34.5% at 40° C. Peak2 Peak2 66.1% 65.5%

Example 10

In Vivo Tests of Complement Inhibition.

UTI-Fc1 (SEQ ID NO:1) affects on the complement system were measured invivo. Female, C3h/HeJ mice were purchased from Jackson Laboratories.Animals are dosed according to the experimental design Table 10. Animalswere injected i.p. (100 ul/mouse) 15 minutes post dose with LPS at timezero. Animals were euthanized at 2 and 4 hours post LPS injection by CO2overdose and blood was collected by cardiac puncture. Blood wastransferred to serum separator microtubes and allowed to clot at roomtemperature for 30 minutes. Subsequently, microtubes were centrifuged at12,000 rpm for 5 minutes and serum was removed and aliquoted to a 96well plate. Rosmarinic acid was used as a positive control and used as 3mg/ml in saline. The 96 well plate was frozen at −20° C. Serum sampleswere analyzed for C5a content by duoset. Statistical significance wasdetermined using Prism graphing software and effects were consideringstatistically significant if p<0.05. As shown in FIG. 10, SEQ ID NO:1(UTI-Fc1) significantly reduced C5a at 20, 50, and 100 mg/kg at 4 hourspost LPS dose.

TABLE 10 Experimental Design (UTI-Fc1 is SEQ ID: 1) Volume LPS DoseConc. (ml/kg) Stimulation conc. Group Description (mg/kg) (mg/ml) RouteLPS (i.p) (mg/ml) Animals 1 naive 8 2 Veh - 2 hr — — 10 iv 30 ug 0.3 8 3UTI-Fc1 - 2 hr 50 5.0 10 iv 30 ug 0.3 8 4 Ros_2 hr_30 mpk 30 3.0 10 sc30 ug 0.3 8 5 Veh - 4 hr — — 10 iv 30 ug 0.3 8 6 UTI-Fc1 - 4 hr_5 mpk 50.5 10 iv 30 ug 0.3 8 7 UTI-Fc1 - 4 hr_20 mpk 20 2.0 10 iv 30 ug 0.3 8 8UTI-Fc1 - 4 hr_50 mpk 50 5.0 10 iv 30 ug 0.3 8 9 UTI-Fc1 - 4 hr_100 mpk100 10.0 10 iv 30 ug 0.3 8 10 Ros_4 hr_30 mpk 30 3.0 10 sc 30 ug 0.3 8

SEQUENCE LISTING SEQ ID NO: 1 UTI-Fc 1 Protein SequenceAVLPQEEEGSGGGQLVTEVTKKEDSCQLGYSAGPCMGMTSRYFYNGTSMACETFQYGGCMGNGNNFVTEKECLQTCRTVAACNLPIVRGPCRAFIQLWAFDAVKGKCVLFPYGGCQGNGNKFYSEKECREYCGVPGDGDEELLGSGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSEQ ID NO: 2 UTI-Fc 1 DNA SequenceGCTGTGCTGCCTCAGGAAGAGGAAGGCTCTGGCGGAGGCCAGCTCGTGACCGAAGTGACCAAGAAAGAGGACTCCTGCCAGCTGGGCTACTCTGCCGGCCCTTGTATGGGCATGACCTCCCGGTACTTCTACAACGGCACCTCCATGGCCTGCGAGACATTCCAGTACGGCGGCTGCATGGGCAACGGCAACAACTTTGTGACAGAGAAAGAGTGCCTGCAGACCTGCAGAACCGTGGCCGCCTGTAACCTGCCTATCGTGCGGGGACCCTGTCGGGCCTTTATCCAGCTGTGGGCCTTCGACGCCGTGAAGGGCAAATGCGTGCTGTTCCCCTATGGCGGCTGCCAGGGAAATGGAAACAAGTTCTACTCCGAGAAAGAATGCCGCGAGTACTGTGGCGTGCCAGGCGACGGGGATGAGGAACTGCTGGGATCAGGCGGCGGAGGCGACAAGACCCATACCTGTCCACCTTGCCCTGCCCCCGAGCTGCTGGGAGGACCTTCTGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCCGGGAAGAGATGACAAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAGGGATTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCSEQ ID NO: 3 UTI-Fc IgG1 3Ser Protein sequenceavlpqeeegsgggqlvtevtkkedscqlgysagpcmgmtsryfyngtsmacetfqyggcmgngnnfvtekeclqtcrtvaacnlpivrgperafiqlwafdavkgkcvlfpyggcqgngnkfysekecreycgvpgdgdeellrepkssdkthtcppcpapellggssvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpasiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgSEQ ID NO: 4 UTI-Fc IgG1 3Ser DNA sequencegctgtgctgcctcaggaagaggaaggctctggcggaggccagctcgtgaccgaagtgaccaagaaagaggactcctgccagctgggctactctgccggcccttgtatgggcatgacctcccggtacttctacaacggcacctccatggcctgcgagacattccagtacggcggctgcatgggcaacggcaacaactttgtgacagagaaagagtgcctgcagacctgcagaaccgtggccgcctgtaacctgcctatcgtgcggggaccctgtcgggcctttatccagctgtgggccttcgacgccgtgaagggcaaatgcgtgctgttcccctatggcggctgccagggaaatggaaacaagttctactccgagaaagaatgccgcgagtactgtggcgtgccaggcgacggggatgaggaactgctgcgggagcccaaatcttccgacaagacccatacctgtccaccttgccctgcccccgagctgctgggaggatcctctgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccctgaagtgacctgcgtggtggtggatgtgtcccacgaggatcccgaagtgaagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagtacaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggattggctgaacggcaaagagtacaagtgcaaggtgtccaacaaggccctgcctgcctccatcgaaaagaccatctccaaggccaagggccagccccgggaaccccaggtgtacacactgccccctagccgggaagagatgacaaagaaccaggtgtccctgacctgtctcgtgaagggattctacccctccgatatcgccgtggaatgggagtccaacggccagcctgagaacaactacaagaccaccccccctgtgctggactccgacggctcattcttcctgtactccaagctgacagtggacaagtcccggtggcagcagggcaacgtgttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccccggcSEQ ID NO: 5 UTI-Fc IgG2 Ser Protein sequenceavlpqeeegsgggqlvtevtkkedscqlgysagpcmgmtsryfyngtsmacetfqyggcmgngnnfvtekeclqtcrtvaacnlpivrgperafiqlwafdavkgkcvlfpyggcqgngnkfysekecreycgvpgdgdeellrkscvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgmevhnaktkpreeqfnstfrvvsyltvvhqdwingkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqks1s1spgkSEQ ID NO: 6 UTI-Fc IgG2 Ser DNA sequencegctgtgctgcctcaggaagaggaaggctctggcggaggccagctcgtgaccgaagtgaccaagaaagaggactcctgccagctgggctactctgccggcccttgtatgggcatgacctcccggtacttctacaacggcacctccatggcctgcgagacattccagtacggcggctgcatgggcaacggcaacaactttgtgacagagaaagagtgcctgcagacctgcagaaccgtggccgcctgtaacctgcctatcgtgcggggaccctgtcgggcctttatccagctgtgggccttcgacgccgtgaagggcaaatgcgtgctgttcccctatggcggctgccagggaaatggaaacaagttctactccgagaaagaatgccgcgagtactgtggcgtgccaggcgacggggatgaggaactgctgcggaaatcctgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcatggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgtcgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctccgggtaaaSEQ ID NO: 7 UTI-Fc IgG2 Protein sequenceavlpqeeegsgggqlvtevtkkedscqlgysagpcmgmtsryfyngtsmacetfqyggcmgngnnfvtekeclqtcrtvaacnlpivrgperafiqlwafdavkgkcvlfpyggcqgngnkfysekecreycgvpgdgdeellrkccvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgmevhnaktkpreeqfnstfrvvsvltvvhqdwlngkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqks1s1spgkSEQ ID NO: 8 UTI-Fc IgG2 DNA sequencegctgtgctgcctcaggaagaggaaggctctggcggaggccagctcgtgaccgaagtgaccaagaaagaggactcctgccagctgggctactctgccggcccttgtatgggcatgacctcccggtacttctacaacggcacctccatggcctgcgagacattccagtacggcggctgcatgggcaacggcaacaactttgtgacagagaaagagtgcctgcagacctgcagaaccgtggccgcctgtaacctgcctatcgtgcggggaccctgtcgggcctttatccagctgtgggccttcgacgccgtgaagggcaaatgcgtgctgttcccctatggcggctgccagggaaatggaaacaagttctactccgagaaagaatgccgcgagtactgtggcgtgccaggcgacggggatgaggaactgctgcggaaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcatggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgtcgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctccgggtaaaSEQ ID NO: 9 UTI-Fc IgG1 3Ser S10A Protein sequenceavlpqeeegagggqlvtevtkkedscqlgysagpcmgmtsryfyngtsmacetfqyggcmgngnnfvtekeclqtcrtvaacnlpivrgperafiqlwafdavkgkcvlfpyggcqgngnkfysekecreycgvpgdgdeellrepkssdkthtcppcpapellggssvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpasiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgSEQ ID NO: 10 UTI-Fc IgG1 3Ser S10A DNA sequencegctgtgctgcctcaggaagaggaaggcgcaggcggaggccagctcgtgaccgaagtgaccaagaaagaggactcctgccagctgggctactctgccggcccttgtatgggcatgacctcccggtacttctacaacggcacctccatggcctgcgagacattccagtacggcggctgcatgggcaacggcaacaactttgtgacagagaaagagtgcctgcagacctgcagaaccgtggccgcctgtaacctgcctatcgtgcggggaccctgtcgggcctttatccagctgtgggccttcgacgccgtgaagggcaaatgcgtgctgttcccctatggcggctgccagggaaatggaaacaagttctactccgagaaagaatgccgcgagtactgtggcgtgccaggcgacggggatgaggaactgctgcgggagcccaaatcttccgacaagacccatacctgtccaccttgccctgcccccgagctgctgggaggatcctctgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccctgaagtgacctgcgtggtggtggatgtgtcccacgaggatcccgaagtgaagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagtacaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggattggctgaacggcaaagagtacaagtgcaaggtgtccaacaaggccctgcctgcctccatcgaaaagaccatctccaaggccaagggccagccccgggaaccccaggtgtacacactgccccctagccgggaagagatgacaaagaaccaggtgtccctgacctgtctcgtgaagggattctacccctccgatatcgccgtggaatgggagtccaacggccagcctgagaacaactacaagaccaccccccctgtgctggactccgacggctcattcttcctgtactccaagctgacagtggacaagtcccggtggcagcagggcaacgtgttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccccggcSEQ ID NO: 11 UTI-Fc IgG1 3Ser m2 Protein sequenceedscqlgysagpcmgmtsryfyngtsmacetfqyggcmgngnnfvtekeclqtcrtvaacnlpivrgperafiqlwafdavkgkcvlfpyggcqgngnkfysekecreycgvpgdgdeellrepkssdkthtcppcpapellggssvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsyltvlhqdwingkeykckvsnkalpasiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspg SEQ ID NO: 12 UTI-Fc IgG1 3Ser m2 DNA sequencegaggactcctgccagctgggctactctgccggcccttgtatgggcatgacctcccggtacttctacaacggcacctccatggcctgcgagacattccagtacggcggctgcatgggcaacggcaacaactttgtgacagagaaagagtgcctgcagacctgcagaaccgtggccgcctgtaacctgcctatcgtgcggggaccctgtcgggcctttatccagctgtgggccttcgacgccgtgaagggcaaatgcgtgctgttcccctatggcggctgccagggaaatggaaacaagttctactccgagaaagaatgccgcgagtactgtggcgtgccaggcgacggggatgaggaactgctgcgggagcccaaatcttccgacaagacccatacctgtccaccttgccctgcccccgagctgctgggaggatcctctgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccctgaagtgacctgcgtggtggtggatgtgtcccacgaggatcccgaagtgaagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagtacaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggattggctgaacggcaaagagtacaagtgcaaggtgtccaacaaggccctgcctgcctccatcgaaaagaccatctccaaggccaagggccagccccgggaaccccaggtgtacacactgccccctagccgggaagagatgacaaagaaccaggtgtccctgacctgtctcgtgaagggattctacccctccgatatcgccgtggaatgggagtccaacggccagcctgagaacaactacaagaccaccccccctgtgctggactccgacggctcattcttcctgtactccaagctgacagtggacaagtcccggtggcagcagggcaacgtgttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccccggc SEQ ID NO: 13 UTI-Fc IgG1 3Ser m1 Protein sequenceavlpqeeegsgggqlvtevtkkepkssdkthtcppcpapellggssvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpasiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgSEQ ID NO: 14 UTI-Fc IgG1 35er m1 DNA sequencegctgtgctgcctcaggaagaggaaggctctggcggaggccagctcgtgaccgaagtgaccaagaaagagcccaaatcttccgacaagacccatacctgtccaccttgccctgcccccgagctgctgggaggatcctctgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccctgaagtgacctgcgtggtggtggatgtgtcccacgaggatcccgaagtgaagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagtacaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggattggctgaacggcaaagagtacaagtgcaaggtgtccaacaaggccctgcctgcctccatcgaaaagaccatctccaaggccaagggccagccccgggaaccccaggtgtacacactgccccctagccgggaagagatgacaaagaaccaggtgtccctgacctgtctcgtgaagggattctacccctccgatatcgccgtggaatgggagtccaacggccagcctgagaacaactacaagaccaccccccctgtgctggactccgacggctcattcttcctgtactccaagctgacagtggacaagtcccggtggcagcagggcaacgtgttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccccggcSEQ ID NO: 15 UTI-Fc IgG1 35er link3 Protein sequenceavlpqeeegsgggqlvtevtkkedscqlgysagpcmgmtsryfyngtsmacetfqyggcmgngnnfvtekeclqtcrtvaacnlpivrgperafiqlwafdavkgkcvlfpyggcqgngnkfysekecreycgvpgdgdeellggggsggggsepkssdkthtcppcpapellggssvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsyltvlhqdwlngkeykckvsnkalpasiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgSEQ ID NO: 16 UTI-Fc IgG1 3Ser link3 DNA sequencegctgtgctgcctcaggaagaggaaggctctggcggaggccagctcgtgaccgaagtgaccaagaaagaggactcctgccagctgggctactctgccggcccttgtatgggcatgacctcccggtacttctacaacggcacctccatggcctgcgagacattccagtacggcggctgcatgggcaacggcaacaactttgtgacagagaaagagtgcctgcagacctgcagaaccgtggccgcctgtaacctgcctatcgtgcggggaccctgtcgggcctttatccagctgtgggccttcgacgccgtgaagggcaaatgcgtgctgttcccctatggcggctgccagggaaatggaaacaagttctactccgagaaagaatgccgcgagtactgtggcgtgccaggcgacggggatgaggaactgctgggaggtggtggatcaggtggcggaggatcagagcccaaatcttccgacaagacccatacctgtccaccttgccctgcccccgagctgctgggaggatcctctgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccctgaagtgacctgcgtggtggtggatgtgtcccacgaggatcccgaagtgaagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagtacaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggattggctgaacggcaaagagtacaagtgcaaggtgtccaacaaggccctgcctgcctccatcgaaaagaccatctccaaggccaagggccagccccgggaaccccaggtgtacacactgccccctagccgggaagagatgacaaagaaccaggtgtccctgacctgtctcgtgaagggattctacccctccgatatcgccgtggaatgggagtccaacggccagcctgagaacaactacaagaccaccccccctgtgctggactccgacggctcattcttcctgtactccaagctgacagtggacaagtcccggtggcagcagggcaacgtgttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccccggcSEQ ID NO: 17 UTI-Fc IgG1 3Ser link2 Protein sequenceavlpqeeegsgggqlvtevtkkedscqlgysagpcmgmtsryfyngtsmacetfqyggcmgngnnfvtekeclqtcrtvaacnlpivrgperafiqlwafdavkgkcvlfpyggcqgngnkfysekecreycgvpgdgdeellrcppcpapellggssvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpasiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgSEQ ID NO: 18 UTI-Fc IgG1 3Ser link2 DNA sequencegctgtgctgcctcaggaagaggaaggctctggcggaggccagctcgtgaccgaagtgaccaagaaagaggactcctgccagctgggctactctgccggcccttgtatgggcatgacctcccggtacttctacaacggcacctccatggcctgcgagacattccagtacggcggctgcatgggcaacggcaacaactttgtgacagagaaagagtgcctgcagacctgcagaaccgtggccgcctgtaacctgcctatcgtgcggggaccctgtcgggcctttatccagctgtgggccttcgacgccgtgaagggcaaatgcgtgctgttcccctatggcggctgccagggaaatggaaacaagttctactccgagaaagaatgccgcgagtactgtggcgtgccaggcgacggggatgaggaactgctgcggtgtccaccttgccctgcccccgagctgctgggaggatcctctgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccctgaagtgacctgcgtggtggtggatgtgtcccacgaggatcccgaagtgaagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagtacaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggattggctgaacggcaaagagtacaagtgcaaggtgtccaacaaggccctgcctgcctccatcgaaaagaccatctccaaggccaagggccagccccgggaaccccaggtgtacacactgccccctagccgggaagagatgacaaagaaccaggtgtccctgacctgtctcgtgaagggattctacccctccgatatcgccgtggaatgggagtccaacggccagcctgagaacaactacaagaccaccccccctgtgctggactccgacggctcattcttcctgtactccaagctgacagtggacaagtcccggtggcagcagggcaacgtgttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccccggcSEQ ID NO: 19 UTI-Fc IgG1 3Ser link1 Protein sequenceavlpqeeegsgggqlvtevtkkedscqlgysagpcmgmtsryfyngtsmacetfqyggcmgngnnfvtekeclqtcrtvaacnlpivrgperafiqlwafdavkgkcvlfpyggcqgngnkfysekecreycgvssvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpasiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspg SEQ ID NO: 20 UTI-Fc IgG1 3Ser link1 DNA sequencegctgtgctgcctcaggaagaggaaggctctggcggaggccagctcgtgaccgaagtgaccaagaaagaggactcctgccagctgggctactctgccggcccttgtatgggcatgacctcccggtacttctacaacggcacctccatggcctgcgagacattccagtacggcggctgcatgggcaacggcaacaactngtgacagagaaagagtgcctgcagacctgcagaaccgtggccgcctgtaacctgcctatcgtgcggggaccctgtcgggcctttatccagctgtgggccttcgacgccgtgaagggcaaatgcgtgctgttcccctatggcggctgccagggaaatggaaacaagttctactccgagaaagaatgccgcgagtactgtggcgtgtcctctgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccctgaagtgacctgcgtggtggtggatgtgtcccacgaggatcccgaagtgaagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagtacaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggattggctgaacggcaaagagtacaagtgcaaggtgtccaacaaggccctgcctgcctccatcgaaaagaccatctccaaggccaagggccagccccgggaaccccaggtgtacacactgccccctagccgggaagagatgacaaagaaccaggtgtccctgacctgtctcgtgaagggattctacccctccgatatcgccgtggaatgggagtccaacggccagcctgagaacaactacaagaccaccccccctgtgctggactccgacggctcattcttcctgtactccaagctgacagtggacaagtcccggtggcagcagggcaacgtgttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccccggcSEQ ID NO: 21 UTI-Fc IgG1 35er K215 1(225 Protein sequenceavlpqeeegsgggqlvtevtssedscqlgysagpcmgmtsryfyngtsmacetfqyggcmgngnnfvtekeclqtcrtvaacnlpivrgperafiqlwafdavkgkcvlfpyggcqgngnkfysekecreycgvpgdgdeellrepkssdkthtcppcpapellggssvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsyltvlhqdwingkeykckvsnkalpasiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgSEQ ID NO: 22 UTI-Fc IgG1 3Ser K21S K22S DNA sequencegctgtgctgcctcaggaagaggaaggctctggcggaggccagctcgtgaccgaagtgacctcctccgaggactcctgccagctgggctactctgccggcccttgtatgggcatgacctcccggtacttctacaacggcacctccatggcctgcgagacattccagtacggcggctgcatgggcaacggcaacaactngtgacagagaaagagtgcctgcagacctgcagaaccgtggccgcctgtaacctgcctatcgtgcggggaccctgtcgggcctttatccagctgtgggccttcgacgccgtgaagggcaaatgcgtgctgttcccctatggcggctgccagggaaatggaaacaagnctactccgagaaagaatgccgcgagtactgtggcgtgccaggcgacggggatgaggaactgctgcgggagcccaaatcttccgacaagacccatacctgtccaccttgccctgcccccgagctgctgggaggatcctctgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccctgaagtgacctgcgtggtggtggatgtgtcccacgaggatcccgaagtgaagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagtacaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggattggctgaacggcaaagagtacaagtgcaaggtgtccaacaaggccctgcctgcctccatcgaaaagaccatctccaaggccaagggccagccccgggaaccccaggtgtacacactgccccctagccgggaagagatgacaaagaaccaggtgtccctgacctgtctcgtgaagggattctacccctccgatatcgccgtggaatgggagtccaacggccagcctgagaacaactacaagaccaccccccctgtgctggactccgacggctcattcttcctgtactccaagctgacagtggacaagtcccggtggcagcagggcaacgtgactcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccccggcSEQ ID NO: 23 UTI-Fc IgG1 3Ser d2 Protein sequenceavlpqeeegsgggqlvtevtkktvaacnlpivrgperafiqlwafdavkgkcvlfpyggcqgngnkfysekecreycgvpgdgdeellrepkssdkthtcppcpapellggssvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpasiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgSEQ ID NO: 24 UTI-Fc IgG1 3Ser d2 DNA sequencegctgtgctgcctcaggaagaggaaggctctggcggaggccagctcgtgaccgaagtgaccaagaaaaccgtggccgcctgtaacctgcctatcgtgcggggaccctgtcgggccthatccagctgtgggccttcgacgccgtgaagggcaaatgcgtgctgttcccctatggcggctgccagggaaatggaaacaagttctactccgagaaagaatgccgcgagtactgtggcgtgccaggcgacggggatgaggaactgctgcgggagcccaaatcttccgacaagacccatacctgtccaccttgccctgcccccgagctgctgggaggatcctctgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccctgaagtgacctgcgtggtggtggatgtgtcccacgaggatcccgaagtgaagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagtacaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggattggctgaacggcaaagagtacaagtgcaaggtgtccaacaaggccctgcctgcctccatcgaaaagaccatctccaaggccaagggccagccccgggaaccccaggtgtacacactgccccctagccgggaagagatgacaaagaaccaggtgtccctgacctgtctcgtgaagggattctacccctccgatatcgccgtggaatgggagtccaacggccagcctgagaacaactacaagaccaccccccctgtgctggactccgacggctcattcttcctgtactccaagctgacagtggacaagtcccggtggcagcagggcaacgtgactcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccccggc SEQ ID NO: 25 UTI-Fc IgG1 3Ser d1 Protein sequenceavlpqeeegsgggqlvtevtkkedscqlgysagpcmgmtsryfyngtsmacetfqyggcmgngnnfvtekeclqtcrepkssdkthtcppcpapellggssvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpasiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgSEQ ID NO: 26 UTI-Fc IgG1 3Ser d1 DNA sequencegctgtgctgcctcaggaagaggaaggctctggcggaggccagctcgtgaccgaagtgaccaagaaagaggactcctgccagctgggctactctgccggcccttgtatgggcatgacctcccggtacttctacaacggcacctccatggcctgcgagacattccagtacggcggctgcatgggcaacggcaacaacthgtgacagagaaagagtgcctgcagacctgcagagagcccaaatcttccgacaagacccatacctgtccaccttgccctgcccccgagctgctgggaggatcctctgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccctgaagtgacctgcgtggtggtggatgtgtcccacgaggatcccgaagtgaagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagtacaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggattggctgaacggcaaagagtacaagtgcaaggtgtccaacaaggccctgcctgcctccatcgaaaagaccatctccaaggccaagggccagccccgggaaccccaggtgtacacactgccccctagccgggaagagatgacaaagaaccaggtgtccctgacctgtctcgtgaagggattctacccctccgatatcgccgtggaatgggagtccaacggccagcctgagaacaactacaagaccaccccccctgtgctggactccgacggctcattcttcctgtactccaagctgacagtggacaagtcccggtggcagcagggcaacgtgttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccccggcSEQ ID NO: 27 mUTI-mFc mIgG1 Protein sequenceavlpqesegsgteplitgfikkedscqlnysegpclgmqeryyyngasmacetfqyggclgngnnfisekdclqtcrtiaacnlpivqgperafiklwafdaaqgkciqfhyggckgngnkfysekeckeycgvpgdgyeelirskivprdcgckpcictvpevssvfifppkpkdvltifitpkvtcvvvdiskddpevqfswfvddvevhtaqtqpreeqfnstfrsyselpimhqdwingkefkcrynsaafpapiektisktkgrpkapqvytipppkeqmakdkvsltcmitdffpeditvewqwngqpaenykntqpimdtdgsyfvysklnvqksnweagntftcsvlheglhnhhtekslshspgkSEQ ID NO: 28 mUTI-mFc mIgG1 DNA sequencegcagtgctgccccaagagagtgaggggtcagggactgagccactaataactgggaccctcaagaaagaagactcctgccagctcaattactcagaaggcccctgcctagggatgcaagagaggtattactacaacggcgcttccatggcctgcgagacctttcaatatgggggttgcctaggcaacggcaacaacttcatctctgagaaggactgtctgcagacatgtcggaccatagcggcctgcaatctccccatagtccaaggcccctgccgagccttcataaagctctgggcatttgatgcagcacaagggaagtgcatccaattccactacgggggctgcaaaggcaacggcaacaaattctactctgagaaggaatgcaaagagtactgtggagtccctggtgatgggtacgaggaactaatacgcagtaaaatcgtgcctcgggactgcggctgcaagccctgcatctgcaccgtgcccgaggtgtcctccgtgttcatcttcccacccaagcccaaggacgtgctgaccatcaccctgacccccaaagtgacctgcgtggtggtggacatctccaaggacgaccccgaggtgcagttcagttggttcgtggacgacgtggaagtgcacaccgcccagacccagcccagagaggaacagttcaactccaccttcagatccgtgtccgagctgcccatcatgcaccaggactggctgaacggcaaagagttcaagtgcagagtgaactccgccgccttcccagcccccatcgaaaagaccatctccaagaccaagggcagacccaaggccccccaggtgtacaccatccccccacccaaagaacagatggccaaggacaaggtgtccctgacctgcatgatcaccgatttcttcccagaggacatcaccgtggaatggcagtggaacggccagcccgccgagaactacaagaacacccagcccatcatggacaccgacggctcctacttcgtgtactccaagctgaacgtgcagaagtccaactgggaggccggcaacaccttcacctgtagcgtgctgcacgagggcctgcacaaccaccacaccgagaagtccctgtcccactcccccggcaagSEQ ID NO: 29 Fc IgG1 3Ser UTI Protein sequenceepkssdkthtcppcpapellggssvflfppkpkdilmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpasiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkggggsggggsggggsavlpqeeegsgggqlvtevtkkedscqlgysagpcmgmtsryfyngtsmacetfqyggcmgngnnfvtekeclqtcrtvaacnlpivrgperafiqlwafdavkgkcvlfpyggcqgngnkfysekecreycgvpgdgdeellrSEQ ID NO: 30 Fc IgG1 3Ser UTI DNA sequencegagcccaaatcttccgacaagacccatacctgtccaccttgccctgcccccgagctgctgggaggatcctctgtgttcctgttccccccaaagcccaaggacaccctgatgatctcccggacccctgaagtgacctgcgtggtggtggatgtgtcccacgaggatcccgaagtgaagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagtacaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggattggctgaacggcaaagagtacaagtgcaaggtgtccaacaaggccctgcctgcctccatcgaaaagaccatctccaaggccaagggccagccccgggaaccccaggtgtacacactgccccctagccgggaagagatgacaaagaaccaggtgtccctgacctgtctcgtgaagggattctacccctccgatatcgccgtggaatgggagtccaacggccagcctgagaacaactacaagaccaccccccctgtgctggactccgacggctcattcttcctgtactccaagctgacagtggacaagtcccggtggcagcagggcaacgtgttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccccggcaagggaggtggtggatcaggaggtggaggttccggtggcggaggatcagctgtgctgcctcaggaagaggaaggctctggcggaggccagctcgtgaccgaagtgaccaagaaagaggactcctgccagctgggctactctgccggcccttgtatgggcatgacctcccggtacttctacaacggcacctccatggcctgcgagacattccagtacggcggctgcatgggcaacggcaacaactttgtgacagagaaagagtgcctgcagacctgcagaaccgtggccgcctgtaacctgcctatcgtgcggggaccctgtcgggcctttatccagctgtgggccttcgacgccgtgaagggcaaatgcgtgctgttcccctatggcggctgccagggaaatggaaacaagttctactccgagaaagaatgccgcgagtactgtggcgtgccaggcgacggggatgaggaactgctgcggSEQ ID NO: 31 hUTI protein sequenceavlpq eeegsgggql vtevtkkeds cqlgysagpcmgmtsryfyn gtsmacetfq yggcmgngnn fvtekeclqt crtvaacnip ivrgperafiqlwafdavkg kcvlfpyggc qgngnkfyse kecreycgvp gdgdeellrf snSEQ ID NO: 32 AMBP preproprotein sequencemrslgallll lsaclavsag pvptppdniq vqenfnisri ygkwynlaig stcpwlkkimdrmtvstlvl gegateaeis mtstrwrkgv ceetsgayek tdtdgkflyh kskwnitmesyvvhtnydey aifltkkfsr hhgptitakl ygrapqlret llqdfrvvaq gvgipedsiftmadrgecvp geqepepili prvrravlpq eeegsgggql vtevtkkeds cqlgysagpcmgmtsryfyn gtsmacetfq yggcmgngnn fvtekeclqt crtvaacnlp ivrgperafiqlwafdavkg kcvlfpyggc qgngnkfyse kecreycgvp gdgdeellrf sn SEQ ID NO: 33SGGGGS SEQ ID NO: 34 SGGGGSGGGGS SEQ ID NO: 35 SGGGGSGGGGSGGGGSSEQ ID NO: 36 SGGGGSGGGGSGGGGSGGGGS SEQ ID NO: 37SGGGGSGGGGSGGGGSGGGGSGGGGS SEQ ID NO: 38 SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSSEQ ID NO: 39 SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS SEQ ID NO: 40SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS SEQ ID NO: 41SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS SEQ ID NO: 42SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS SEQ ID NO: 43GSGGGSGGGGSGGGGS

1-9. (canceled)
 10. An isolated human urinary trypsin inhibitor (hUTI)fusion protein comprising: (a) hUTI or a hUTI variant that retains hUTIfunctional activity, (b) a flexible peptide linker (L) selected from thegroup consisting of SEQ ID Nos: 33-42, and (c) an IgG Fc domain thatlacks the first constant region immunoglobulin domain, or fragmentthereof that retains wild type IgG Fc functional activity.
 11. The hUTIfusion protein of claim 10, wherein the hUTI fusion protein demonstratesgreater thermal stability than the UTI fusion proteins described inChinese patent application CN103044554A.
 12. The hUTI fusion protein ofclaim 10, wherein the hUTI variant is a hUTI fragment.
 13. The hUTIfusion protein of claim 10, wherein the hUTI variant is a hUTI analogue.14. The hUTI fusion protein of claim 10, wherein the Fc domain binds toan Fc receptor on a human cell.
 15. The hUTI fusion protein of claim 10,wherein the Fc domain is an analogue of an Fc domain.
 16. The hUTIfusion protein of claim 10, wherein the Fc domain is a fragment of an Fcdomain.
 17. The hUTI fusion protein of claim 10, further comprisingsignal peptide MGWSCIILFLVATATGVHS (SEQ ID NO:49).
 18. The hUTI fusionprotein of claim 10, wherein the hUTI fusion protein is a monomer. 19.The hUTI fusion protein of claim 10, wherein the hUTI fusion protein isa multimer.
 20. The hUTI fusion protein of claim 10, wherein the hUTIfusion protein is a dimer.
 21. The hUTI fusion protein of claim 19 or20, wherein the multimer or dimer comprises polypeptide chains that areassociated covalently.
 22. The hUTI fusion protein of claim 19 or 20,wherein the multimer or dimer comprises polypeptide chains that areassociated non-covalently.
 23. The hUTI fusion protein of claim 10,wherein the hUTI fusion protein comprises an amino acid sequenceselected from the group consisting of SEQ ID Nos: 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, and
 29. 24. The hUTI fusion protein of claim10, wherein the hUTI protein is a variant human UTI protein that retainshUTI functional activity.
 25. A dimer comprising two isolated hUTIfusion proteins of claim 10, wherein the Fc domains, or fragmentsthereof, are associated covalently.
 26. A pharmaceutical compositioncomprising the UTI fusion protein of claim 10 or the dimer of claim 25,and a pharmaceutically acceptable excipient.
 27. A nucleic acid encodingthe UTI fusion protein of any one of claim 10 or the dimer of claim 25.28. An expression vector comprising the nucleic acid of claim
 27. 29. Arecombinant host cell comprising the expression vector of claim
 28. 30.The recombinant host cell of claim 29, wherein the cell is selected fromthe group consisting of a mammalian cell, an insect cell, an E. colicell, a yeast cell, and a plant cell.
 31. The recombinant host cell ofclaim 30, wherein the mammalian cell is selected from the groupconsisting of a Chinese hamster ovary (CHO) cell, an HEK 293 cell, anNSO cell, a HeLa cell, a baby hamster kidney (BHK) cell, a monkey kidneycell (COS) and a human hepatocellular carcinoma cell.
 32. A method ofproducing a hUTI fusion protein, the method comprising the step ofplacing the recombinant host cell of claim 31 in a growth medium suchthat a-recombinant fusion protein is expressed, and isolating therecombinant fusion protein from the cell or growth medium.
 33. A methodof producing a UTI fusion protein, wherein the UTI fusion protein ofclaim 10 or the dimer of claim 25, is produced in a transgenic animal.34. The isolated UTI fusion protein of claim 10 or the dimer of claim25, wherein the Fc domain is selected from the group consisting of anIgG1, an IgG2 and an IgG4 Fc domain.
 35. The isolated UTI fusion proteinof claim 10 or the dimer of claim 25, wherein the Fc domain is an IgG1Fc domain.
 36. A method of treating a UTI-related condition comprisingadministering to a patient in need thereof an effective amount of theUTI fusion protein of claim 10 or the dimer of claim
 25. 37. The methodof claim 36, wherein the UTI-related condition is selected from thegroup consisting of pancreatitis, endoscopy-induced pancreatitis, acutepancreatitis, arthritis, severe acute respiratory syndrome, systemicinflammatory response syndrome, acute circulatory failure, sepsis,hepatitis, appendicitis, colitis, organ failure, organ damage, pancreasdamage, kidney damage, lung damage, reperfusion injury, Stevens-Johnsonsyndrome, toxic epidermal necrolysis, shock, ischemic injury, acute lunginjury, lung injury caused by acute aortic dissection, asthma, lunginflammation, pneumonia, ventilator-associated pneumonia, disseminatedintravascular coagulation, and acute respiratory distress syndrome. 38.The method of claim 36, wherein the UTI-related condition is acutepancreatitis.